Interpretive Summary: Viroids, the smallest pathogens yet described, are tiny, circular single-stranded RNA molecules that encode no proteins. Yet, like the much larger viruses, are able to replicate autonomously in infected host plants. Viroids cause a number of economically important diseases, but, in a crop like citrus, the dwarfing that is usually associated with viroid infection can actually be beneficial. One possibility is to develop improved citrus viroid dwarfing agents suitable for use under subtropical conditions. In order to develop the technology necessary to isolate stable viroid strains having the desired biological properties, a series of trial experiments was carried out using potato spindle tuber viroid, a "model" viroid that also infects tomato. We found that only a minority (approximately 5%) of all mutants generated by sequence randomization were infectious. The results of our experiments will be of greatest interest to (i) other molecular biologists interested in viroid/virus evolution at the molecular level and (ii) horticulturists intersted in increasing citrus production efficiency.

Technical Abstract:
Sequence variation among naturally occurring strains of Potato spindle tuber viroid (PSTVd) is concentrated in two widely separated domains of its rod-like secondary structure. Single point mutations in the pathogenicity domain have a dramatic effect on symptom expression, and the spontaneous in vivo conversion of the type strain PSTVd-Intermediate to the rapidly replicating, severe variant RG1 required only three substitutions [Gruner et al. (1995) Virology 209, 60-69]. To identify possible evolutionary pathways linking these two stably-replicating variants, we randomized five positions in an infectious cDNA copy of PSTVd-Intermediate and screened the resulting population of 768 sequences for viable variants. Twenty four variants were recovered, and subsequent numerical simulations indicated the actual number of viable variants to be <50. Spontaneous mutations were detected at nearby positions in the pathogenicity domain but not the variable domain, suggesting that sequence evolution proceeds independently in these two portions of the molecule. No naturally occurring PSTVd variants were recovered, and only one viable variant linking PSTVd-Intermediate to RG1 was found among the mutagenesis progeny. The overall pattern of sequence changes observed indicates that PSTVd-Int occupies a comparatively steep fitness peak within the overall variant population.